jum/DIAGNOSTIC NUCLEAR MEDICINE

ENDOTOXIN AS A CAUSE OF ASEPTIC AFTER RADIONUCLIDE CISTERNOGRApHy

JamesF. Cooper and John C. Harbert The University of Tennessee Center for the Health Sciences, Memphis, Tennessee, and Georgetown University Hospital, Washington, D.C.

The role of pyrogens in aseptic meningitis after radionuclide cisternography was studied by TABLE 1. ADVERSE REACTIONS FOLLOWING means of the Limulus test, a sensitive detector RADIONUCLIDECISTERNOGRApHy of endotoxin. During a 15-month period, 39 Radiooctivetracer'Symptomatic reactions associated with cisternography were with reference meningitist

reported. Ten samples of specific lots of the 1―I-IHSA Detmerand Blacker(1) radioactive drugs implicated in 20 of these re “l-IHSA Nicol (2) actions were tested and all reacted strongly “l-IHSA Oldham and Staab (3) 1311-IHSA Jonasand Braunstein(4) positive to the Limulus test. The less sensitive ‘°mTc-HSAand rabbit pyrogen test was negative for these prepa 1―l-IHSA Barnesand Fish(5) 5/50(10%) rations when tested on a dose-per-weight basis. ‘311-IHSA Messed and Rieder (6) 7/50 (14/.) ‘31l-IHSA Atkins, et al (7) 19 Our findings apparently provide clinical evi “ln-DTPA Aldersonand Siegel (8) 2/130 (1.5°!.) dence for the observation made in animals that “11n-DTPA Wagner, et al (9) 1/16(6%) endotoxin is at least 1,000 times more toxic * IHSA,radioiodinated(1311)humanserumalbumin,USP; intrathecally than intravenously. The data impli HSA, human serum albumin; DTPA, diethylenetriamine pen taaceticacid, cate endotoxin contamination as a cause of ad t Numberof patientswith symptomaticmeningitis/total verse reactions to radionuclide cisternography. number of patients in study, if reported. We conclude that the USP pyrogen test is insuffi ciently sensitive for intrathecal injectables and should be supplemented by the Limulus test. following intrathecal injection of ‘31I-IHSA (7). Adverse reactions after cisternography occurred at Georgetown Hospital in Washington, D.C., and led Radioiodinated human serum albumin (1311-IHSA) us to study their cause (11). @ and the DTPA chelates of 11n and 169Th have be Although the etiology of these adverse reactions come accepted as tracers for radionuclide cisternog was unknown, the amount of albumin (1—3,12), raphy because of their reliability in demonstrating chemical irritants (1 ,5,8) , sensitization (1 ,8) , and cerebrospinal fluid dynamics. Nevertheless, appre pyrogens (5,8,10) were advanced as possiblecauses. hension attends the use of this procedure because of The advent of the Limulus test made available a isolated reports of aseptic meningitis following intra rapid, sensitive method for detecting bacterial endo thecal ‘311-IHSAinjections (1—4). Alarmingly high toxin (pyrogens) in radiopharmaceuticals and other reaction rates, up to 14% (Table 1) , were reported parenterals (13—15). By means of the Limulus test, in recent studies, and reactions to one of the newer endotoxin was detected in radiopharmaceuticals, agents were reported (5—9). Banerji and Spencer which caused aseptic meningitis after intrathecal in (10) reported that 27% of 60 patients had a febrile jection, and this contaminant was implicated as the response of at least 1°Fwithin 24 hr of ‘311-IHSA etiologic agent in the adverse reactions reported here. cisternography; when patients had accompanying pneumoencephalograms, the incidence was 41 % of Received Jan. 30, 1975; revision accepted April 10, 1975. 88 patients. The survey of adverse reactions to radio For reprints contact: J. F. Cooper, Radiomedications Sec pharmaceuticals sponsored by the Society of Nuclear tion, Dept. of Medicinal Chemistry, The University of Ten nesseeCenter for the Health Sciences, 26 S. Dunlap, Room Medicine presented 19 reports of aseptic meningitis 107,Memphis,Tenn. 38163.

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MATERIALSANDMETHODS 0 and V grades are positive for bacterial endo Glassware was cleaned, sterilized, and rendered toxin. It has been our experience with endotoxin pyrogen-free by heating in a dry-air oven at 200°C solutions and small-volume parenteral products that for at least 2 hr. Sterile, pyrogen-free solutions were a positive Limulus test with a gel time greater than used for all procedures. Specimens of Limulus poly 25 mm will not produce a pyrogenic response in phemus (horseshoe crab) were collected near Chin rabbits when the same solution is injected intrave coteague, Va., and Limulus amebocyte lysate was nously at a dose of I ml/kg (13,14). On this assump prepared by a modification of the Levin method tion, positive reactions for radiopharmaceuticals are (14,15) . The amebocytes were lysed by adding Ster categorized as follows by the gel time (the time in ile Water for Injection, USP, to cells in a 2 :1 ratio minutes required for a firm gel to form after prompt by volume while agitating vigorously. The super mixing and incubation of the assay mixture) : strong nate was removed and stored at 40°C. positive, gel time less than 30 mm; positive, gel time 30—60mm; and trace, gel time greater than 60 mm Purified endotoxins of Escherichia coli (E. coli or a V grade test result. 026 :B6, Difco No. 5745 14) and Kiebsiella pneu Preparations of 1311-IHSA, radioiodmated (131!) moniae, the FDA reference pyrogen ( 16 ) , were pre human serum albumin, USP, were the products of pared in stock solutions of 100 ,@g/ml. The sensitivity Abbott Laboratories (Risa-131-H) and E. R. Squibb of lysate preparations was determined with freshly & Sons (Albumotope-1311). Preparations of 1@In prepared twofold dilutions of these endotoxins, 2 diethylenetriamine pentaacetic acid (@In-DTPA) through 0.1 25 ng/ml concentrations. Acceptable were obtained from one supplier. lysate detected 1 ng/ml of these endotoxins within 1 hr by forming a firm gel. Sodium Chloride for In jection, USP (Abbott Laboratories), or Sterile Water RESULTS for Injection, USP, was used for endotoxin stock Limulus tests were conducted on over 100 differ solutions and dilutions and served as the negative ent commercial lots of ‘311-IHSAand 1111n-DTPA control when mixed with Limulus amebocyte lysate. preparations during a 15-month period beginning in Test units of lyophilized lysate were prepared by March 1972. Test samples and notifications of ad lyophilizing 0.1 ml of tris-buffered lysate (pH 7) in verse reactions came to us from investigators and 2-ml funnel-top ampuls (Wheaton Glass Co. ) and industry or by response to a published request for sealing with rubber closures under vacuum. The participation in this study (17). Of 39 cases of lyophilized lysate was stored below 0°Cor at 4°C aseptic meningitis, we obtained 10 samples of radio and assayed as described above. A slight increase pharmaceuticals for assay implicated in 20 adverse in sensitivity was often observed for the lyophilized patient reactions. Every lot of 1311-IHSA and 1111n- units. This was attributed to the fact that the relative DTPA tested associated with an adverse reaction endotoxin concentration was higher in the test am yielded a strongly positive result by the Limulus test. puls when reconstituted with the test solution than One reaction occurred after Risa-1 3 1-H intra when mixed in equal parts with liquid lysate. A com thecal injection but the specific lot was unavailable mercial Limulus amebocyte lysate (Pyrogent, by for a Limulus test. However, 35 of the lots of this Mallinckrodt, Inc. ) was used as directed by the drug tested by the Limulus test during this period package insert in a comparative study. were either negative or contained trace levels of The Limulus test was conducted by injecting 0.25W- endotoxin. 0.3 ml of the radiopharmaceutical directly into a Of 10 lots of Albumotope-131I associated with 24 test ampul of lyophilized lysate. The assay mixture reactions, 5 lots were obtained that were implicated was thoroughly and quickly mixed and then incu in 8 adverse reactions. All five reacted strongly posi bated immediately in a water bath at 37°C. Test tive with the Limulus test (Table 2). Another lot ampuls were observed periodically without further tested during this time was also strongly positive. agitation for at least 60 mm for the formation of an One lot of Albumotope-1311 (assay date of July opaque gel. The time required for gel formation 21 , 1972, Table 2), which was both Limulus-test was recorded in minutes as the time of gelation. The positive and associated with untoward effects, was results were graded in our laboratory as follows: assayed after serial dilutions to approximate the G, formation of a firm gel that remains intact when endotoxin concentration. The undiluted solution and inverted slowly to 180°; V, increase in viscosity, 1 : 10 and 1 : 100 dilutions (in Sodium Chloride for opacity, and observance of gelatinous granules; and Injection, USP) gave 12-, 15-, and 25-mm gel times, N, negative,no formation of opaque gelatinousgran respectively, by the Limulus test. These results mdi ules. cated a high level of endotoxin contamination in the

810 JOURNAL OF NUCLEAR MEDICINE DIAGNOSTIC NUCLEAR MEDICINE

10 ,@g/ml. Therefore, inadequate column washing TABLE 2. ADVERSE REACTIONS TO could have contributed significant amounts of endo RADIONUCLIDE CISTERNOGRAPHYAND toxin to these radioiodinated drugs. LIMULUS TEST RESULTSFOR ALBUMOTOPE.131l* We learned of 14 reactions to the ‘‘‘In-DTPA AdversereactionsAssay preparations. Five were isolated events, but nine dote for lot CasesHospitalsLimulus testst reactions followed the use of two specific lots in May 1973. The manufacturer supplied samples of these April 28, 1972 2 2 NA June 2, 1972 2 2 NA two lots and all solutions associated with their formu June 9, 1972 2 G-12 lation as well as three other implicated lots. All five July 14, 1972 G-12 lots of “‘In-DTPAwere found positive by the Limu July 21, 1972 2 2 G-1 2 July 28, 1972 7 2 NA lus test. By testing the solutions used in formulation, August 4, 1972 4 2 NA the source of pyrogenic contamination was traced to September 1, 1972 G-1 8 a phosphate buffer. Eleven other lots of ‘“In-DTPA September 22, 1972 2 G-20 October 18, 1972 None reported G-20 were negative by the Limulus test. October 25, 1972 NA Cultures of the ‘311-IHSAand ‘‘In-DTPAlots

. Radioiodinated (“I) human serum albumin USP. were negative. Also, when sample volume permitted, t G, positive resultwith time (mm)required for formation the commercial Limulus amebocyte lysate was also of a firm gel of Limulus amebocyte lysate after prompt mix ing and incubation of the assay mixture. NA, not available used to test these radiopharmaceuticals; test results for Limulus test. were essentiallythe same.

DISCUSSION original sample, i.e., greater than 0. 1 @g/ml when compared to purified endotoxins (see MATERIALS Adverse reactions to radionuclide cisternography, AND METHODS). usually described as aseptic or chemical meningitis, A second lot of Albumotope-1311 (assay date of have been reported as mild to serious (1—9). Recov September 1, 1972, Table 2) yielded a strongly posi ery was usually rapid with no residual sequelae. All tive Limulus test : a gel time of I 8 mm. The rabbit cultures of cerebrospinal fluid and the implicated pyrogen test (0.14 mI/kg), conducted at the time radiopharmaceutical were negative. of release by the supplier, was negative with a 0.32°C In the first two reported reactions, 28 mg albumin mean temperature increase; a 0.5 °Cmean tempera (‘311-IHSA) was administered intrathecally to one ture increase was defined as a positive pyrogen test patient and 100—130 mg to the second (1 ,2) . Di for a biologic at the time of this study. Two months Chiro, et al (12) suggested that the total amount later this lot was retested at a greater dose ( 1 mI/kg) of albumin administered be limited to 4 mg because and produced a positive result, a 0.83°C mean tem their experience with high specific activity ‘311-IHSA perature increase in three rabbits. (over 200 patients) and DomTc@HSA(40 patients) The Albumotope-1311 lots discussed above ( 1% was without untoward reaction. Subsequently, re albumin) were prepared by radioiodination of a 25% actions were reported with the high specific activity human serum albumin, Hyland Lot 0490R015A. 131I4H5A and “‘In-DTPA,when it became avail A Limulus test of the parent lot of human serum able, with similar clinical and laboratory findings albumin (25% ) was positive with a gel time of 40 (8). Therefore, albumin apparently was not the mm, but a tenfold dilution of the same lot was nega single causative factor. @ tive. Also, the rabbit pyrogen test ( 3 ml/kg) of the The two commercial preparations of I-IHSA 25 % human serum albumin lot was negative. The used in cisternography (not an FDA-approved use) parent lot of concentrated human serum albumin contain 1% albumin, benzyl alcohol as a preserva was not a significant source of contamination. Low tive, and a buffer system to maintain neutrality. The level endotoxin-like contamination is not uncommon intrathecal toxicity of benzyl alcohol in dogs was in these albumin products (14). studied by DeLand; he found no evidence of menin During preparation, 1311-IHSA is passed through geal reaction following cisterna magna injections in a washed anion-exchange resin column to reduce the concentrations up to ten times that found in this amount of nonprotein-bound radioiodine. Since the product (18). Although the product formulations resin was suspected as a source of contamination, were similar, the adverse reactions to ‘31I-IHSAre an initial resin column wash was supplied by one ported in this study followed the use of one supplier's manufacturer for Limulus testing. Serial dilutions to product in 24 of 25 cases. The 24 adverse reactions 1 : 10,000 gave positive results, which indicated that to Albumotope-'31 (Table 2) occurred during a the initial wash contained endotoxin greater than 7-month period. Since all six samples of the radio

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active drug that were produced during this period intrathecally administered bacterial endotoxin. These reacted similarly to the Limulus test (gel times of data also demonstrate that the USP pyrogen test is 20 mm or less) , there may have been endotoxin not sufficiently sensitive to serve as a screening test contamination in all the lots prepared by the manu for intrathecal drugs. facturer during the 7-month period. Initial studies of the relative sensitivities of the A likely source of pyrogenic contamination for rabbit and Limulus tests for purified pyrogen dem I @ the 131I-IHSA preparations was the anion-exchange onstrated that the Limulus test was five to ten times resin used in production for radiochemical punfica more sensitive when its endpoint was formation of tion. Resin washes provided by one manufacturer a firm gel within I hr and the rabbit pyrogen test were highly contaminated according to Limulus test dose of 1 ml/kg produced a minimal average in results. This reinforces the necessity to wash ion crease in rectal temperature of 0.5 °Cin three or exchange resins copiously with pyrogen-free solu more rabbits (13) . Elm, et al (23) confirmed this tions. observation in a study of three bacterial endotoxins Barnes, et al (5) and DiChiro (12) have demon using similar criteria. strated that stringent quality-contrOl measures for The supplier's intravenous pyrogen test dose to preparing intrathecal radiotracers are effective in re rabbits was 0.14 ml/kg for 1311-IHSAand 1 ml/ ducing aseptic meningitis following radionuclide cis rabbit for the @In-DTPA preparation. - The total ternography. After the manufacturers employed the human dose was usually less than 1 ml administered Limulus test in their quality-control program, no intrathecally. Pyrogen testing of parenteral products reactions to intrathecal 1311-IHSA were reported to on a dose-per-weight basis has been validated by the Society of Nuclear Medicine Registry of Adverse Greisman, et al (24) but only for intravenous ad Reactions in 1973 (19). ministration. Since the pyrogen test (25) allows small We suggest that endotoxin or an endotoxin-like volume parenterals to be tested relative to the hu contaminant was the cause of most, if not all, of the man dose, it is common practice in the drug industry adverse reactions reported here. The evidence sup to build a safety factor into the rabbit pyrogen test porting this contention may be summarized as fol dose. For example, the human dose for this pro lows : (A) the Limulus test detected endotoxin in cedure was assumed to be 1 ml 1311-IHSA/70-kg all ten available lots of radiotracers that were im man (0.014 mi/kg) ; therefore, the 0.14-mi/kg rab plicated in meningeal reactions; (B) a single source bit pyrogen test dose described above provided a of ‘311-IHSAwas associated with 24 of 25 reactions safety factor of 10. By the same reasoning, a 1-mi/kg although both formulations were similar; (C) menin pyrogen test dose would have a safety factor of 70. geal reactions followed the use of two different The Limulus test would have a safety factor of 350 radiotracers (1311-IHSA and @1In-DTPA); (D) for for a 1-ml dose of 1311-IHSAin adults. Since endo one Limulus-positive 1311-IHSA lot, a repeat pyro toxin is apparently more potent by the intrathecai gen test, at a higher dose, was pyrogenic in rabbits; route, we believe that a Limulus test should be re (E) endotoxin contamination was traced to produc quired for intrathecal drugs to serve as a more sensi tion components; and (F) to date quality-assurance tive test and provide a higher safety factor. measures have reduced the incidence of this adverse Since the gelation of lysate by endotoxin in the reaction. Limulus test is dependent on the concentration of The preparations that were positive for endotoxin bacterial endotoxin (13,15) , more than qualitative by the Limulus test had passed the supplier's pyrogen information may be gained from a Limulus test. An and sterility tests. Endotoxin administered intrave estimate of the pyrogenic level may be made by nously induces host cells (granulocytes, Kupifer cells, comparing the gel time or serial dilution assays of and others) to release endogenous pyrogen that acts the test sample with purified endotoxins, i.e., iipo on the thermoregulatory center in the hypothalamus polysaccharides from the cell wall of gram-negative to increase body temperature (20) . However, the bacteria. fate of intrathecally administered pyrogen and its Several substances other than endotoxin have been mechanism of meningeal irritation is unknown. Ben reported to induce gelation of Limulus amebocyte nett, et at (21 ) demonstrated that endotoxin in the lysate. Elm, et al (23) found that synthetic poly subarachnoid space of dogs and rabbits was at least nucleotides gelled lysate; however, we were unable I ,000 times more potent than the intravenous to reproduce these results using commercial Poly route in producing febrile response. These findings (1)-poly (C). They also reported that certain throm were reproduced in cats (22) . Our data may be the boactive materials gelled lysate but the presence of first clinical evidence of the increased potency of endotoxin contamination in these drugs was not ruled

812 JOURNALOF NUCLEARMEDICINE DIAGNOSTIC NUCLEAR MEDICINE out (23 ) . Microgram levels of isolated peptidogly ing 1111n-DTPA cisternography. I Nuci Med 14: 609—611, cans from gram-positive bacteria produced positive 1973 Limulus tests but intact gram-positive organisms 9. WAGNERMS, MORTONRJ, GRAHAMIS, et al: Quan titative cisternography with “In-DTPA. I Nucl Med 14: failed to induce gelation (26). Only endotoxin has 462, 1973 been reported to induce gelation at nanogram con 10. BANERJIMA, SPENCERRP: Febrile response to cere centrations; therefore, it is unlikely that any other brospinal fluid flow studies. I Nucl Med 13: 655, 1972 contaminant could have produced the biologic re 11. COOPERJF, HARBERTJC: Bacterial endotoxin as a sponses and positive Limulus tests reported here cause of aseptic meningitis following radionuclide cisternog raphy.I Nuci Med 14: 387, 1973 (13,15,23,26). 12. DICrnR0 G, ASHBURN WL, BRINER WH: Techne When the Limulus test was used clinically to de tium Tc 99m serum albumin for cisternography. Arch tect endotoxin in other bodily fluids, these test re Neurol 19:218—227,1968 suits correlated well with clinical and bacteriologic 13. COOPERJF, LEvn'i J, WAGNER HN: Quantitative findings (27,28) . This study demonstrates the use comparison of in vitro and in vivo methods for the detec fulness of the Limulus test to detect endotoxin in tion of endotoxin. I Lab Clin Med 78: 138—148, 1971 14. COOPERJF, HOCHSTEINHD, SELIGMANNEB: The drugs for which the rabbit pyrogen test is either Limulustest for endotoxin (pyrogen) in radiopharmaceuti insufficiently sensitive or otherwise unsuitable. Al cals and biologicals. Bull Parenter Drug Assoc 26: 153—162, though the Limulus test is unofficial at this time, we 1972 conclude that it should supplement the rabbit test is. LEVINJ, BANGFB: Clottableproteinin Limulus: in screening intrathecal drugs for pyrogen. Its localization and kinetics of its by endotoxin. Thromb Diath Haemorrh 19: 186—197,1968 ACKNOWLEDGMENTS 16. CASTORGB, KANTORN, KNOLL E, et al: Charac teristics of a highly purified pyrogenic . We are grateful to the physicians who provided samples I Pharm Sci60: 1578—1580,1971 for Limulus testing and clinical information on adverse 17. HARBERT JC, COOPERJ: Aseptic meningitis following reactions, and to Hal Atkins, who supplied data from isotope cisternography. I Nuci Med 13: 778, 1972 the Society of Nuclear Medicine Registry of Adverse Reac 18. DELAND FH: Intrathecal toxicity studies with benzyl tions. We are also grateful to individuals in the radiophar alcohol.Toxicol AppI Pharmacol 25: 153—156,1973 maceutical industry and the FDA's Bureau of Biologics who 19. ATKINS HL: Adverse reaction registry. SNM News supplied information regarding the radiopharmaceuticals letter 5: 6, 1974 discussed here. 20. ATKINS E, BODEL P: . N EngI I Med 286: This work was presented in part at the 20th Annual Meet 27—34,1972 ing of the Society of Nuclear Medicine in June 1973. We 21. BENNETr IL, PETERSDORFRG, KEENE WR: Patho thank Ben Friedman and Jack Levin for reviewing this genesis of fever: Evidence for direct cerebral action of bac manuscript. terial endotoxins. Trans Assoc Am Physicians 70: 64—73, REFERENCES I957 22. VILLABLANCAJ, MYERS RD: Fever produced by mi 1. DETMERDE, BLACKERHM: A case of aseptic menin croinjection of typhoid vaccine into hypothalamus of cats. gitis secondary to intrathecal injection of I'°human serum Am I Physiol 208: 703—707,1965 albumin. Neurology 15: 642—643, 1965 23. ELIN RJ, WOLFF SM : Nonspecificity of the Limulus 2. NICOL CF: A second case of aseptic meningitis fol amebocyte lysate test : Positive reactions with polynucleotides lowing isotope cisternography using Ii―human serum albu and proteins.I InfectDis 128: 349—352,1973 mm. Neurology 17: 199—200,1967 24. GREISMANSE, HORNICKRB: Comparative pyrogenic 3. OLDHAMRK, STAABEV: Aseptic meningitis following reactivity of rabbit and man to bacterial endotoxin. Proc the intrathecal injection of radioiodinated serum albumin. Soc Erp BiolMed 131: 1154—1158,1969 Radiology97: 317—321,1970 25. Pharmacopeia of the United States of A merica : Pyro 4. JONASS. BRAUNSTEINP: Neurogenic bladder as a gen test, Bethesda, The United States Pharmacopeial Con complication of isotope cisternography. I Nucl Med 13: 763—764,1972 vention, Inc., 1970, p 886 26. WILDFEUER A, HEYMER B, SCHLEIFER KH, et al: 5. BARNES B, FISH M : Chemical meningitis as a compli Investigations on the specificity of the Limulus test for the cation of isotope cisternography. Neurology 22: 83—91,1972 6. MESSERTB, RIEDERMJ: RISA cisternography:Study detection of endotoxin. AppI Microbiol 28: 867—871,1974 of spinal fluid changes associated with intrathecal RISA in 27. LEVINJ, POORETE, ZAUBERNP, et al: Detection of jection. Neurology 22: 789—792,1972 endotoxin in the of patients with sepsis due to gram 7. ATKINSHL, HAUSERW, RICHARDSP. et al: Adverse negative bacteria. N Engi I Med 283: 1313—1316,1970 reactions to radiopharmaceuticals. I NucI Med 13: 232—233, 28. NACHUM R, LIPSEY A, SIEGEL SE: Rapid detection 1972 of gram-negative bacterial meningitis by the Limulus lysate 8. ALDERSON P0, SIEGEL BA : Adverse reactions follow test. N Engi I Med 289: 931—934,1973

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